Eukaryotic biosensor making use of a calcium regulated light emitting enzyme

ABSTRACT

The present invention provides a method of using eukaryotic cells being transformed with a light emitting Ca2+ regulated photoprotein gene for determining the presence or absence of at least one toxic substance in a sample and for assisting in the identification of the toxicant(s). More specifically there is provided a toxicity assay for various uses including determining the presence of toxins and in particular heavy metals and organophenols, general cytoxicity testing of pure chemicals and chemical mixtures in particular for drug development testing, testing of food and drink products, cosmetics testing and identification of organisms in particular of fungal strains.

The present invention provides a method of using transformed eukaryoticcells or organisms for determining the presence or absence of at leastone toxic substance in a sample and for assisting in the identificationof the toxicant(s). More specifically there is provided a toxicity assayfor various uses including determining the presence of toxins, generalcytotoxicity testing of pure chemicals and chemical mixtures inparticular for drug development testing, testing of food and drinkproducts, cosmetics testing and identification of organisms inparticular of fungal strains

The release of contaminating substances into an environment such as awaterway or an area of agricultural land can have serious effects on theecosystems found in that environment. It is important to be able toanalyse these effects both prior to the release of such contaminants soas to manage their treatment or release, and after release so as todetermine and counteract their effects.

Current methods used to monitor water quality and screen effluentgenerally involve chemical toxicity tests. However, these tests requirea general idea of the type of contaminant being tested for and can bevery expensive.

Similarly the presence of contaminating substances or toxins can beproblematic in other areas such as food and drink manufacture andcosmetics manufacture. There are also instances, such as in drugdevelopment and cosmetic industry, where the substance of interest i.e.the potential new drug may itself be a contaminating substance or toxinand this needs to be checked.

Biosensors are used for toxicity testing and are well known in thefield. Toxicity depends on a variety of factors including pH,temperature, salinity and contaminant concentration, but dependsespecially on the test organism used in the sensor.

One of the most commonly used organisms is the bioluminescent bacterium,Vibrio fischeri. The bioluminescence involved is mediated by theluciferin-luciferase enzyme system wherein light emission is dependenton the electron transfer chain. Any disruption to the electron transferchain, for example on exposure to a toxicant, affects light emission.Light emission at the time a substance is added is therefore indicativeof the presence of a toxic substance.

This system, however, only provides a simple indication of whether acontaminant is toxic or not. No detailed information is obtained on howtoxic the contaminant is, nor is the contaminant identified.

The terms toxicant and toxin as herein described relate to compounds,chemicals and mixtures of chemicals which have an effect on eukaryoticcells or organisms and in particular which are toxic to eukaryoticorganisms such as fungus or which have anti-fungal activity.

The term eukaryote as herein described relates to eukaryotic cells ororganisms.

According to a first aspect of the present invention there is provided amethod of determining the presence of a toxicant in a test sample,comprising the steps of;

-   -   exposing a eukaryote that has been transformed with a light        emitting Ca²⁺ regulated photoprotein gene to a test sample    -   measuring the light produced by the transformed cell/organism    -   determining whether the amount of light is above or below a        defined threshold at the time of exposure.

Optionally the eukaryote is a fungi. (throughout this document fungishould be considered under its typical classification as covering bothmulticellular organisms and unicellular organisms such as the yeastSaccharomyces cerviseae Preferably the fungi is a filamentous fungi.

More preferably the fungi is of the Aspergillus species.

Alternatively the eukaryote is a mammalian cell.

A further alternative is that the eukaryote is a plant cell.

Preferably the test sample comprises a toxicant.

Preferably the light emitting Ca²⁺ regulated photoprotein gene is arecombinant gene.

Preferably the light emitting Ca²⁺ regulated photoprotein gene isselected from the group comprising;

-   -   aequorin gene    -   halistaurin (mitrocomin) gene    -   phialidin (clytin) gene    -   obelin gene    -   mnemiopsin gene    -   berovin gene

Optionally, the light emitting Ca²⁺ regulated photoprotein gene may be afunctional homologue of a gene selected from the group comprising;

-   -   aequorin gene    -   halistaurin (mitrocomin) gene    -   phialidin (clytin) gene    -   obelin gene    -   mnemiopsin gene    -   berovin gene

Most preferably the light emitting Ca²⁺ regulated photoprotein gene isan aequorin gene.

More preferably the light emitting Ca²⁺ regulated photoprotein gene is arecombinant aequorin gene.

Preferably the light that is measured is in the form of luminescence.

Optionally the test sample is added in advance of the application of astimulus to the test sample.

Preferably the stimulus is at least one or more from the groupcomprising; mechanical perturbation, hypo-osmotic shock, and change inexternal calcium chloride concentration, temperature shock, pH shock.

Preferably the test sample is added 1 minute to 1 hour prior to theapplication of the stimulus.

More preferably the test sample is added 5 minutes prior to theapplication of the stimulus.

More preferably the test sample is added 30 minutes prior to theapplication of the stimulus.

According to a second aspect of the present invention there is provideda method of determining the presence of a toxicant in a test sample,comprising the steps of;

-   -   exposing a eukaryote that has been transformed with a light        emitting Ca²⁺ regulated photoprotein gene to a test sample    -   measuring the light produced by the transformed cell/organism    -   determining whether the amount of light is above a defined        threshold at a specified time after the time of exposure.

Optionally the method comprises the step of determining whether theamount of light is below a defined threshold.

Optionally the specified time after the time of exposure is 11 minutes.

Optionally the eukaryote is a fungi.

Preferably the fungi is a filamentous fungi.

More preferably the fungi is of the Aspergillus species.

Alternatively the eukaryote is a mammalian cell.

A further alternative is that the eukaryote is a plant cell.

Preferably the test sample comprises a toxicant.

Preferably the light emitting Ca²⁺ regulated photoprotein gene is arecombinant gene.

Preferably the light emitting Ca²⁺ regulated photoprotein gene isselected from the group comprising;

-   -   aequorin gene    -   halistaurin (mitrocomin) gene    -   phialidin (clytin) gene    -   obelin gene    -   mnemiopsin gene    -   berovin gene

Optionally, the light emitting Ca²⁺ regulated photoprotein gene may be afunctional homologue of a gene selected from the group comprising;

-   -   aequorin gene    -   halistaurin (mitrocomin) gene    -   phialidin (clytin) gene    -   obelin gene    -   mnemiopsin gene    -   berovin gene

Most preferably the light emitting Ca²⁺ regulated photoprotein gene isan aequorin gene.

More preferably the light emitting Ca²⁺ regulated photoprotein gene is arecombinant aequorin gene.

Preferably the light that is measured is in the form of luminescence.

Optionally the test sample is added in advance of the application of astimulus to the test sample.

Preferably the stimulus is at least one or more from the groupcomprising; mechanical perturbation, hypo-osmotic shock, change inexternal calcium chloride concentration, temperature shock, pH shock.

Preferably the test sample is added 1 minute to 1 hour prior to theapplication of the stimulus.

More preferably the test sample is added 5 minutes prior to theapplication of the stimulus.

More preferably the test sample is added 30 minutes prior to theapplication of the stimulus.

According to a third aspect of the present invention there is provided amethod of determining the presence of a toxicant in a test sample,comprising the steps of;

-   -   exposing a eukaryote that has been transformed with a light        emitting Ca²⁺ regulated photoprotein gene to a test sample    -   measuring the light produced by the transformed cell/organism    -   and comparing at least one parameter of the light measurement        data with a bank of known toxicity reference data.

Optionally the method comprises the step of determining whether theamount of light is below a defined threshold.

Optionally the specified time after the time of exposure is 11 minutes.

Optionally the eukaryote is a fungi.

Preferably the fungi is a filamentous fungi.

More preferably the fungi is of the Aspergillus species.

Do we need next two sentences

Most preferably the fungi is Aspergillus awamori.

Most preferably the strain of Aspergillus awamori is strain 66A.

Alternatively the eukaryote is a mammalian cell.

A further alternative is that the eukaryote is a plant cell.

Preferably the test sample comprises a toxicant.

Preferably the light emitting Ca²⁺ regulated photoprotein gene is arecombinant gene.

Preferably the light emitting Ca²⁺ regulated photoprotein gene isselected from the group comprising;

-   -   aequorin gene    -   halistaurin (mitrocomin) gene    -   phialidin (clytin) gene    -   obelin gene    -   mnemiopsin gene    -   berovin gene

Optionally, the light emitting Ca²⁺ regulated photoprotein gene may be afunctional homologue of a gene selected from the group comprising;

-   -   aequorin gene    -   halistaurin (mitrocomin) gene    -   phialidin (clytin) gene    -   obelin gene    -   mnemiopsin gene    -   berovin gene

Most preferably the light emitting Ca²⁺ regulated photoprotein gene isan aequorin gene.

More preferably the light emitting Ca²⁺ regulated photoprotein gene is arecombinant aequorin gene.

Preferably the light that is measured is in the form of luminescence.

Optionally the test sample is added in advance of the application of astimulus to the test sample.

Preferably the stimulus is at least one or more from the groupcomprising; mechanical perturbation, hypo-osmotic shock, change inexternal calcium chloride concentration, temperture shock, pH shock.

Preferably the test sample is added 1 minute to 1 hour prior to theapplication of the stimulus.

More preferably the test sample is added 5 minutes prior to theapplication of the stimulus.

More preferably the test sample is added 30 minutes prior to theapplication of the stimulus.

Preferably, the method is used to determine the amount of toxicant inthe sample.

Optionally, the method is used to identify the toxicant in the sample.

According to a fourth aspect of the present invention there is provideda method of determining the presence of a toxicant in a test sample,comprising the steps of;

-   -   exposing a eukaryote that has been transformed with a light        emitting Ca²⁺ regulated photoprotein gene to a test sample    -   measuring the light produced by the transformed cell/organism    -   converting the light data into a cytosolic free calcium ion        concentration trace,    -   and comparing at least one parameter of the cytosolic free        calcium ion concentration trace with a bank of known toxicity        reference data.

Optionally the method comprises the step of determining whether theamount of light is below a defined threshold.

Optionally the specified time after the time of exposure is 11 minutes.

Optionally the eukaryote is a fungi.

Preferably the fungi is a filamentous fungi.

More preferably the fungi is of the Aspergillus species.

Alternatively the eukaryote is a mammalian cell.

A further alternative is that the eukaryote is a plant cell.

Preferably the test sample comprises a toxicant.

Preferably the light emitting Ca²⁺ regulated photoprotein gene is arecombinant gene.

Preferably the light emitting Ca²⁺ regulated photoprotein gene isselected from the group comprising;

-   -   aequorin gene    -   halistaurin (mitrocomin) gene    -   phialidin (clytin) gene    -   obelin gene    -   mnemiopsin gene    -   berovin gene

Optionally, the light emitting Ca²⁺ regulated photoprotein gene may be afunctional homologue of a gene selected from the group comprising;

-   -   aequorin gene    -   halistaurin (mitrocomin) gene    -   phialidin (clytin) gene    -   obelin gene    -   mnemiopsin gene    -   berovin gene

Most preferably the light emitting Ca²⁺ regulated photoprotein gene isan aequorin gene.

More preferably the light emitting Ca²⁺ regulated photoprotein gene is arecombinant aequorin gene.

Preferably the light that is measured is in the form of luminescence.

Optionally the test sample is added in advance of the application of astimulus to the test sample.

Preferably the stimulus is at least one or more from the groupcomprising; mechanical perturbation, hypo-osmotic shock, change inexternal calcium chloride concentration, temperature shock, pH shock.

Preferably the test sample is added 1 minute to 1 hour prior to theapplication of the stimulus.

More preferably the test sample is added 5 minutes prior to theapplication of the stimulus.

More preferably the test sample is added 30 minutes prior to theapplication of the stimulus. Preferably light is measured for between 1minute and 5 hours following the application of the stimulus.

More preferably light is measured for 5 minutes following theapplication of the stimulus.

Preferably, the cytosolic free calcium ion trace is a plot of thecytosolic free calcium ion concentration against time.

Preferably the parameter is at least one or more selected from the groupcomprising;

-   -   lag time    -   rise time    -   absolute amplitude    -   relative amplitude    -   Length of transient    -   number of cytosolic free calcium ion concentration increases    -   percentage increase in final cytosolic free calcium ion        concentration resting level    -   percentage increase in recovery time    -   percentage increase in pre-stimulating cytosolic free calcium        ion concentration resting level    -   Total concentration of Ca²⁺ released.

Preferably, the method is used to determine the amount of toxicant inthe sample.

Optionally, the method is used to identify the toxicant in the sample.

According to a fifth aspect of the present invention there is providedan assay for use in determining the presence of a known toxicant in atest sample, the assay comprising the steps of;

-   -   exposing a fungi transformed with a recombinant aequorin gene to        a test sample of a substance,    -   measuring the luminescence produced by the fungi,    -   converting the luminescence data into a cytosolic free calcium        ion concentration trace,    -   and comparing at least one parameter of the cytosolic free        calcium ion concentration trace with a bank of known toxicity        reference data.

Preferably the cytosolic free calcium ion trace is a plot of thecytosolic free calcium ion concentration against time.

Preferably the fungi transformed with a recombinant aequorin gene is afilamentous fungi.

More preferably the fungi is of the Aspergillus species.

Preferably the substance is a contaminant.

Preferably the substance is a contaminated sample.

Preferably the parameter is at least one or more selected from the groupcomprising; lag time, rise time, absolute amplitude, relative amplitude,length of transient at 20%, 50% and 80% of maximum amplitude, number ofcytosolic free calcium ion concentration increases, percentage increasein final cytosolic free calcium ion concentration resting level,percentage increase in recovery time and percentage increase in thetotal amount of Ca²⁺ released.

Optionally, the test sample is added in advance of the application of astimulus to the test sample.

Preferably the stimulus is at least one or more from the groupcomprising; mechanical perturbation, hypo-osmotic shock, change inexternal calcium chloride concentration, temperature shock and pH shock.

Preferably the test sample is added 1 minute to 1 hour prior to theapplication of the stimulus.

More preferably the test sample is added 5 minutes prior to theapplication of the stimulus.

More preferably the test sample is added 30 minutes prior to theapplication of the stimulus.

In such instances, the parameters may include at least one or moreselected from the group comprising; lag time, rise time, absoluteamplitude, relative amplitude Length of transient at 20%, 50% and 80% ofmaximum amplitude, number of cytosolic free calcium ion concentrationincreases, percentage increase in final cytosolic free calcium ionconcentration resting level, percentage increase in recovery time,percentage increase in pre-stimulating cytosolic free calcium ionconcentration resting level and percentage increase in the total amountof Ca²⁺ released.

Preferably luminescence is measured for between 1 minute and 5 hoursfollowing the application of the stimulus.

More preferably luminescence is measured for 5 minutes following theapplication of the stimulus.

Preferably, the method is used to determine the amount of toxicant inthe sample.

Optionally, the method is used to identify the toxicant in the sample.

In order to further explain the present invention details of a number ofexperiments are provided.

A first experiment comprises testing the effect of pre-incubation ofAspergillus awamori with toxicants on cytosolic free calcium ionconcentration response to an increase in external calcium chloride.

A further set of experiments described herein shows attempts to obtaincharacteristic data for a range of different toxicants at a number ofdifferent concentrations. The results demonstrate that each toxicant ateach concentration produces a distinctive cytosolic free calcium ionconcentration trace whose traits could be used to identify andcharacterise a toxicant present in a test sample.

A final experiment attempts to determine whether it is possible toidentify and characterise individual toxicants from testing samples ofmixtures of toxicants in different proportions. The traces produced aredistinct for each mixture.

These results show that it is possible to characterise and identify aspecific toxicant from a test sample by using the characteristic dataobtained from a cytosolic free calcium ion concentration trace.

It is also possible to characterise and identify a specific toxicantfrom a test sample by using the characteristic data obtained from lightreadings. The main difference between doing light emission and cytosolicfree calcium ion concentrations is the removing the step of convertingthe luminescence data into a cytosolic free calcium ion concentrationtrace”.

So the Method is:

An assay for use in determining the presence of a known toxicant in atest sample, the assay comprising the steps of;

-   -   exposing a fungi transformed with a recombinant aequorin gene to        a test sample of a substance,    -   measuring the luminescence produced by the fungi in relative        light units (RLU),    -   and calculating the following parameters: lag time, rise time,        length of transient (LT₂₀, LT₅₀, LT₈₀), absolute amplitude,        relative amplitude, recover time, final level of luminescence,        initial level of luminescence, total luminescence.

Since RLU are not normalised with regard to the biomass, the parametersmeasured in relative light units (RLU) are different from the cytosolicfree calcium ion concentration [Ca²⁺]. FIGS. 24 and 25 show that thedecrease in amplitude caused by 260 mg/l Cr⁶⁺ is 75% in RLU, and only65% in Ca²⁺ concentration. Other parameters would differ in a similarway.

Most of the toxicity testing for environmental pollutants is usuallycarried out using RLU and therefore the light-emitting essay would beparticularly helpful if used alongside other existing biosensors.

The parameters referred to herein relate to the following;

Lag Time, the time from addition of the test sample to the time when thecytosolic free calcium ion concentration, [Ca²⁺]_(c), began to rise;

Rise Time, the time from addition of the test sample to the time atwhich maximum [Ca²⁺]_(c) was reached;

Number of [Ca²⁺]_(c) Rises, the number of peaks in [Ca²⁺]_(c);

Percentage Increase in Final [Ca²⁺ c Resting Level, the percentageincrease in resting [Ca²⁺]_(c) at the end of the experiment, where thecontrol value is taken to be 100%;

Percentage Increase in Recovery Time, percentage increase in recoverytime where recovery time represents the total amount of [Ca²⁺]_(c)released during the period of time from the point when the maximumamplitude following calcium chloride treatment was achieved to the pointwhen the [Ca²⁺]_(c) reached its final resting level. Recovery time wasinitially calculated for control cultures. In the control this period oftime was calculated as 250 seconds. For the cultures subjected to thetreatment with toxicant(s) the total amount of [Ca²⁺]_(c) was calculatedfor the same period of 250 seconds starting from the maximum amplitude.The recovery time of the control cultures was therefore:$\frac{\begin{matrix}{{{total}\quad{amount}\quad{{of}\quad\left\lbrack {Ca}^{2 +} \right\rbrack}_{c}\quad({\mu M})\quad{for}\quad{the}\quad{toxicant}} -} \\{{treated}\quad{samples}\quad{over}\quad 250\quad{seconds} \times 100}\end{matrix}}{\begin{matrix}{{total}\quad{amount}\quad{{of}\quad\left\lbrack {Ca}^{2 +} \right\rbrack}_{c}\quad({\mu M})\quad{for}\quad{the}\quad{control}\quad{sample}} \\{{over}\quad 250\quad{seconds}}\end{matrix}}$

Percentage Increase in pre-Stimulating [Ca²⁺]_(c) Resting Level, thepercentage increase in [Ca²⁺]_(c) prior to the stimulus, where thecontrol value is taken to be 100%.

Percentage change in total amount of calcium released during thetransient at stage 1—calculated by integration of the all luminescenceobtained after addition of the compounds of interest before subsequentstimulation with physico-chemical stimuli.

Percentage change in total amount of calcium released during thetransient at stage 2—calculated by integration of the all luminescenceobtained after the fungus is stimulated with one of the physico-chemicalstimuli.

Percentage change in total amount of calcium released during the wholetransient—calculated by integration of the all luminescence obtainedduring the period of experiment.

Length of transient (LT)—this parameter describes the length of thetransient when the amplitude of the response is equal a certainpercentage from the maximum amplitude.

LT₂₀ (Length of transient at Amplitude=20% of maximum Amplitude)

LT₅₀ (Length of transient at Amplitude=50% of maximum Amplitude)

LT₈₀ (Length of transient at Amplitude=80% of maximum Amplitude)

All secondary increases have to be analysed by the same parameters asprimary increases during stages 1 and 2.

E.g. Amplitude, length, rise time, lag time,

Percentage change in amplitude should be assessed as the absolute valuefrom point 0 (A_(a)) and as the relative value from the initial restinglevel (A_(r)). The relative changes assess the ability of of theeukaryote to respond to the physiological stimuli. This parameter isimportant to assess the physiological state of the eukaryote.

There is also the possibility of combining one or more of theseparameters to obtain further values which can be used for identificationof the toxicants in the mixture. For example, the summation of amplitudeand recovery time will give the value of total cytosolic free calciumions emitted from the time when [Ca²⁺]_(c) reaches its peak. Alsosummation of lag time and rise time will give the total time requiredfor [Ca²⁺]_(c) to reach its peak. The division of final [Ca²⁺]_(c)resting level onto the pre-stimulation [Ca²⁺]_(c) resting level willshow how many times the [Ca²⁺]_(c) resting level has changed afterstimulation. Similarly, a division of the final [Ca²⁺]_(c) resting levelonto the initial [Ca²⁺]_(c) resting level prior to the addition oftoxicant(s) gives further identifying data. Additionally, the summationof all the data points of the trace gives the total amount of cytosolicfree calcium ions released during the monitoring period.

As mammalian cells are more complex than other eukaryotes such as fungior plants typically more parameters will be considered.

The present invention will now be described with reference to thefollowing non-limiting examples and with reference to the figures,wherein:

FIG. 1 shows the characteristic [Ca²⁺]_(c) trace produced on addition of5 mM external CaCl₂, following a 5 minute pre-incubation with differentconcentrations of 3,5-DCP.

FIG. 2 shows the characteristic [Ca²⁺]_(c) trace produced on addition of5 mM external CaCl₂, following a 5 minute pre-incubation with differentconcentrations of Cr⁶⁺.

FIG. 3 shows the characteristic [Ca²⁺]_(c) trace produced on addition of5 mM external CaCl₂, following a 5 minute pre-incubation with differentconcentrations of Zn²⁺.

FIG. 4 shows the characteristic [Ca²⁺]_(c) trace produced on addition of5 mM external CaCl₂, following a 30 minute pre-incubation with differentconcentrations of 3,5-DCP.

FIG. 5 shows the characteristic [Ca²⁺]_(c) trace produced on addition of5 mM external CaCl₂, following a 30 minute pre-incubation with differentconcentrations of Cr⁶⁺.

FIG. 6 shows the characteristic [Ca²⁺]_(c) trace produced on addition of5 mM external CaCl₂, following a 30 minute pre-incubation with differentconcentrations of Zn²⁺.

FIG. 7 shows the characteristic cytosolic free calcium ionconcentration, [Ca²⁺]_(c), trace produced on addition of 5 mM CaCl₂following a 5 minute pre-incubation with different concentrations of3,5-dichlorophenol, 3,5-DCP.

FIG. 8 shows the characteristic [Ca²⁺]_(c) trace produced on addition of5 mM CaCl₂, following a 30 minute pre-incubation with differentconcentrations of 3,5-DCP.

FIG. 9 shows the characteristic [Ca²⁺]_(c) trace produced on addition of5 mM CaCl₂, following a 5 minute pre-incubation with differentconcentrations of chromium ions, Cr⁶⁺.

FIG. 10 shows the characteristic [Ca²⁺]_(c) trace produced on additionof 5 mM CaCl₂, following a 30 minute pre-incubation with differentconcentrations of chromium ions, Cr⁶⁺.

FIG. 11 shows the characteristic [Ca²⁺]_(c) trace produced on additionof 5 mM CaCl₂, following a 5 minute pre-incubation with differentconcentrations of zinc ions, Zn²⁺.

FIG. 12 shows the characteristic [Ca²⁺]_(c) trace produced on additionof 5 mM CaCl₂, following a 30 minute pre-incubation with differentconcentrations of zinc ions, Zn²⁺.

FIG. 13 shows the values of [Ca²⁺]_(c) trace parameters characteristicfor different concentrations of pentochlorophenol, PCP; sodium dodecylsulphate, SDS; and Toluene. Parameters assessed are Lag Time, LT; RiseTime, RT; Amplitude, A; Length of transient, LT50; Percentage Increasein pre-Stimulating [Ca²⁺]_(c) Resting Level, % IpreSRL; PercentageIncrease in Final [Ca²⁺]_(c) Resting Level, % IFRL; Percentage Increasein Recovery Time, % IRT; and Number of [Ca²⁺]_(c) Increases.

FIG. 14 shows the values of [Ca²⁺]_(c) trace parameters characteristicfor 3,5-DCP, PCP, Zn²⁺, Cr⁶⁺, Toluene, and SDS. Parameters assessed areLag Time, LT; Rise Time, RT; Amplitude, A; Length of transient, LT50;Percentage Increase in pre-Stimulating [Ca²⁺]_(c) Resting Level, %IpreSRL; Percentage Increase in Final [Ca²⁺]_(c) Resting Level, % IFRL;Percentage Increase in Recovery Time, % IRT; and Number of [Ca^(2+])_(c) Increases.

FIG. 15 shows the values of [Ca²⁺]_(c) trace parameters characteristicfor different mixtures of toxicants. Parameters assessed are Lag Time,LT; Rise Time, RT; Amplitude, A; Length of transient, LT50; PercentageIncrease in pre-Stimulating [Ca²⁺]_(c) Resting Level, % IpreSRL;Percentage Increase in Final [Ca²⁺]_(c) Resting Level, % IFRL;Percentage Increase in Recovery Time, % IRT; and Number of [Ca²⁺]_(c)Increases.

Effect Of Pre-Incubation of Aspergillus Awamori with Toxicants on[Ca²⁺]_(c) Response to External Calcium Chloride

12 ml of sterile VS medium was inoculated with 1×10⁵ spores per ml A.awamori strain 66A. 100 μl of the inoculated medium was added to eachwell of a 96-well plate and cultured in a humidity chamber in thepresence of free water at 30° C. for 24 hours.

The following toxicants were tested: 3,5-dichlorophenol, zinc sulphate,and potassium dichromate. Each toxicant was added in a total volume of25 μl VS medium or water 5 or 30 minutes before addition of 5 mM calciumchloride.

Luminescence was monitored for 5 minutes following addition of CaCl₂.Aequorin was completely discharged by adding 3M calcium chloride in 20%ethanol. The total concentration is thus 1.5 M calcium chloride in 10%ethanol.

Luminometry was performed using an EG & G Berthold (Bad Wildbad,Germany) LB96P Microlumat luminometer. Luminescence data was convertedfrom real light units to [Ca²⁺]_(c) values using the following equation:PCa=0.332588 (−log k)+5.5593,where k=luminescence counts per second/total luminescence counts. Totalluminescence is measured as an integral of all luminescence up tocomplete aequorin discharge.

The Equation is first described in Fricker, M. D., Plieth, C., Knight,H., Blancaflor, E., Knight, M. R., White, N. S., and Gilroy, S. 1999.Fluorescence and Luminescence Techniques to Probe Ion Activities inLiving Plant Cells. In Mason, W. T., editor, Fluorescent and LuminescentProbes. Academic Press. London. pp. 569-596.

The following parameters were assessed:

Rise Time, Amplitude, Length of transient, LT50 and Final [Ca²⁺]_(c)Resting Level.

Effects of Different Concentrations of Toxicants on [Ca²⁺]_(c) Traces

Aspergillus awamori were transformed with an expression vector(pAEQ1-15) comprising a gene for synthetic apoaequorin (aeqS) under thecontrol of the constitutive glucose-6-phosphate dehydrogenase promoter(gpdA).

These transformants were cultured in 100 μl of Vogel's medium with 1%sucrose (VS medium) in microwell plates for 24 hours before addition ofa toxicant or a control of distilled water. Toxicants were dissolved inwater to give the concentrations shown below. 25 μl of the each of thefollowing concentrations were added to each culture: TOXICANTCONCENTRATIONS (mg/l) 3,5-dichlorophenol (3,5-DCP) 0.112, 11.2, 112Chromium ions (Cr⁶⁺) 15, 120, 260 Zinc ions (Zn²⁺) 180, 350, 700, 1300

The cultures were incubated for 5 or 30 minutes before addition of 100μl 5 mM CaCl₂. Luminescence was measured for 5 minutes using a plateluminometer. Luminescence data was manually converted from relativelight units to cytosolic free calcium ion concentration, [Ca²⁺]_(c).This was then plotted against time and parameters of this trace wereanalysed. Parameters assessed were as follows:

Rise Time, the time from addition of CaCl₂ to the moment when maximum[Ca²⁺]_(c) was achieved;

Amplitude, the maximum [Ca²⁺]_(c) reached during the experiment;

Length of transient, at 50% of maximum amplitude the width of thetransient at the point where the amplitude equals half of the maximumamplitude of the transient;

and Final Resting [Ca²⁺]_(c) Level, the resting [Ca²⁺]_(c) at the end ofthe experiment.

Effects of Further Toxicants on [Ca²⁺]_(c) Traces

Cultures of Aspergillus awamori as described above were used to test theeffects of further toxicants. The concentrations of toxicants testedwere made up as follows in water, where the concentrations tested werebased on Dutch target and intervention values for toxicants and KellyGuidelines for the classification of contaminated soils: TOXICANTCONCENTRATION (mg/l) Pentochlorophenol, PCP 0.01, 0.1, 1, 5, 10 Sodiumdodecyl sulphate, SDS 1, 10, 50, 100, 500 Toluene 1, 25 3,5-DCP 10 Zn²⁺700 Cr⁶⁺ 15

In the first set-up (S1), 100 μl of each toxicant concentration or ofthe control (VS medium) were added to the cultures through built-ininjectors and luminescence monitored for 5 minutes. In a second set ofexperiments (S2), cultures were pre-incubated with the toxicant orcontrol for 5 minutes before addition of 5 mM CaCl₂ in a total volume of25 μl distilled water (pre-incubation can be anywhere between 1 minuteand 96 hours). Luminescence was monitored for 5 minutes followingaddition of CaCl₂. (monitoring can be anywhere between 1 minute and 96hours). Luminescence data was converted from relative light units to[Ca²⁺]_(c) values as described above. The following parameters wereassessed in S1:

Lag Time, the time from addition of CaCl₂ to the time when [Ca²⁺]_(c)began to rise;

Rise Time;

Absolute amplitude;

Relative amplitude

Length of transient (LT20, LT50, LT80);

Percentage Increase in Final [Ca²⁺]_(c) Resting Level, where the controlvalue was taken to be 100%;

Percentage Increase in Recovery Time, where the control value was takento be 100%; and Number of [Ca²⁺]_(c) Increases, the number of [Ca²⁺]_(c)transients.

Total Ca²⁺ concentration

In S2, the Percentage Increase in pre-Stimulating [Ca²⁺]_(c) RestingLevel, where the control value was taken to be 100%, was assessed inaddition to all of the parameters tested in S1.

Effects of Mixtures Containing Different Propotion of Toxicants on[Ca²⁺]_(c) Traces

The experiments described when examining the effects of furthertoxicants were repeated for different mixtures of toxicants. Thefollowing mixtures were made up in water for testing:

6 mg/l 3,5-DCP+12 mg/l Cr⁶⁺

30 mg/l Cr⁶⁺+350 mg/l Zn²⁺

10 mg/l 3,5-DCP+350 mg/l Zn²⁺

6 mg/l 3,5-DCP+12 mg/l Cr⁶⁺+350 mg/l Zn²⁺

Mixture 1: 20 mg/l Cadmium

-   -   100 mg/l Copper    -   50 mg/l Chromium    -   250 mg/l Zinc    -   500 mg/l SDS

Mixture 2: 20 mg/l Cadmium

-   -   100 mg/l Copper    -   50 mg/l Chromium    -   250 mg/l Zinc

These experiments demonstrate a novel finding that each toxicant resultsin a different and characteristic [Ca²⁺]_(c) transient. Additionallyeach concentration of toxicant produces a unique [Ca²⁺]_(c) transient.From these characteristic fingerprint responses a profile of data can bebuilt up and used to create a bank of data for each toxicant. Resultsfrom testing samples can be compared with this data bank and thepresence of a particular toxicant can thus be determined. Furthermore,details such as the mode of action of the toxicant, and the amount oftoxicant present can be deduced from a comparison with the bank ofpre-gathered data.

Examples of Types of Testing that can be Carried out According to thePresent Invention

Specific examples of types of test that can be carried out according tothe present invention are given below. Although the tests below describethe use of aequorin expressed fungi according to the present invention,it can be seen that any appropriate eukaryotic cell or organism could beused (i.e. mammalian cells in place of the fungi) which has beentransformed with any appropriate gene according to the present invention(i.e. halistaurin in place of aequorin)

The examples refer to the following figures in which;

FIG. 16 shows a graph indicating the effect of 6 environmental sampleson [Ca²⁺]_(c);

FIG. 17 shows a graph indicating the effect of ibuprofen analogue on[Ca²⁺]_(c);

FIG. 18 shows a graph indicating the effect of verpamil on [Ca²⁺]_(c);

FIG. 19 is a table summarising the profiles of the ibuprofen™((S)-(−)-o-Acetulmandelic acid) and verapamil™ (Verapamil hydrochloride)analogues;

FIG. 20 is a table summarising profiles of cyclopiazonic acid (CPA) andKP4 (mycotoxin produced by Ustilago spp);

FIG. 21 is a graph showing the dose-dependent effect of KP4 on the[Ca²⁺]_(c) response to 5 mM external CaCl₂ (results represent mean±SE);

FIG. 22 a is a graph showing the effect of known antifungal drugs on[Ca²⁺]_(c) in Aspergillus nidulans;

FIG. 22 b is a graph showing the effect of known antifungal drugs on[Ca²⁺]_(c) in Aspergillus niger;

FIG. 22 c is a graph showing the effect of known antifungal drugs on[Ca²⁺]_(c) in Aspergillus awamori; and

FIG. 23 is a graph showing the effect of amphotericin B on [Ca²⁺]_(c)(results represent mean±SE).

FIG. 24 shows a graph showing the ffect of Cr⁶⁺ (5 min preincubation) onaequorin light emission in response to the addition of external CaCl₂ (5mM). Results represent mean±SE.

FIG. 25 shows a graph showing the effect of Cr⁶⁺ (5 min preincubation)on [Ca²⁺]_(c) in response to the addition of external CaCl₂ (5 mM).Results represent mean±SE.

General Cytotoxicity

pure chemicals and chemical mixtures can be tested for their toxicityusing aequorin-expressed fungi. Procedure:

-   -   i. Add compound(s) of interest to fungus    -   ii. Monitor [Ca²⁺]_(c) for 5 min    -   iii. Then stimulate fungus with mechanical perturbation,        hypo-osmotic, hyper-osmotic shock    -   iv. Monitor [Ca²⁺]_(c) for further 5 min

The parameter to be assessed is [Ca²⁺]_(c) final resting level. If[Ca²⁺]_(c) resting level is still elevated more then 50% after the 11min measurements the compound(s) are toxic. The level of toxicity can beassessed by subsequent monitoring of [Ca²⁺]_(c) for several hours. Thelonger the [Ca²⁺]_(c) concentration is out of normal the more toxic thecompound(s) are. This way there is no need for complicated software andthis type of approach is ideally suitable for binary answer, based on 1parameter.

FIG. 16 shows a graph indicating the effect of 6 environmental sampleson [Ca²⁺]_(c). The graph indicates that sample 006 is toxic as the[Ca²⁺]_(c) final resting level is increased by more than 150% comparedwith the control.

Another parameter for the analysis of general toxicity is the totalamount of [Ca²⁺]_(c) emitted. Based on this parameter it is very easy tobuild dose response curves (see FIG. 21).

High Information Multiparameters Analysis

In cases when the binary answer is not sufficient aequorin-basedbiosensor can produce much more detailed data characterising not onlythe general cytotoxicity but also penetrability (by analysing the timebetween administration of the compound to the point when [Ca²⁺]_(c)starts to increase) and modes-of-action of the compounds (by comparingthe profile of [Ca²⁺]_(c) changes of the compound(s) of interest to thelibrary of profiles). If the mode-of-action of the compound(s) ofinterest is unique and unknown than the present invention can suggestwhether the compound(s) causes the permeabilization of the membrane,opening of ion channels or the alteration in behaviour of Ca²⁺ carriers.This approach is ideally suitable for analysis of combinations ofcompounds.

This approach can be used for both pollutants monitoring as previouslydescribed but also for the analysis of drug toxicity. e.g. ibuprofen andverapamil. FIGS. 17 and 18 show the effect of ibuprofen and verapamilanalogues on the [Ca²⁺]_(c) and the table shown in FIG. 19 furthersummarises the profiles of the ibuprofen and verapamil analogues.

Profiling Compounds of Interest and Creating the Libraries ofFingerprints of Compounds

The present invention is ideally suitable for creating the library ofprofiles for certain substances. These profiles are unique to a compoundwith the particular mode-of-action. Also they are unique to the strainof fungus used, which allows creating very details and reproduciblefingerprint of a particular compound using the present invention. Theprofiles can be created with different physico-chemical stimuli (e.g.mechanical perturbation, hypo-osmotic, hyper-osmotic shock, cold shock,heat shock, pH shock). These fingerprints can be programmed into thesoftware and any compounds or mixtures of interest can be screened tomatch the desired fingerprint.

Procedure to create the fingerprint:

-   -   Monitor initial [Ca²⁺]_(c) resting level for 1 min    -   Add compound(s) of interest to fungus    -   Monitor [Ca²⁺]_(c) for 5 min    -   Then stimulate fungus with mechanical perturbation,        hypo-osmotic, hyper-osmotic shock    -   Monitor [Ca²⁺]_(c) for further 5 min    -   Based on the data obtained the following parameters can be        quantified for each [Ca²⁺]_(c) increase occurring during the        experiment.        -   Lag time        -   Rise time        -   Amplitude absolute        -   Amplitude relative        -   Length of transient (LT₂₀, LT₅₀, LT₈₀)        -   Initial [Ca²⁺]_(c) level        -   Final [Ca²⁺]_(c) resting level        -   Recovery time        -   Total concentration of [Ca²⁺]_(c)    -   Above 6 steps can be performed on different strains    -   Compound can be tested at different concentrations

Considering the nature of the experiment the minimum number ofparameters produced by one compound at a particular concentration on onefungal strain is equal 22.

Analysis of Food and Drink Products for the Presence of Mycotoxins

Fungi transformed with aequorin gene can be also used for the analysisof food and drink products for the presence of mycotoxins since thesetoxins affect [Ca²⁺]_(c). Examples of such effects are shown in FIG. 20where the effects of cyclopiazonic acid (CPA) and KP4 (mycotoxinsproduced by Ustilago spp) are summarised.

Cosmetics Safety Testing

Since EU regulations forbid the use of animal testing for cosmeticsindustry the manufacturers are looking at the alternative methods toassess the effect of new products. As the present invention is ideallysuited for analysis of not only pure compounds but also their mixtures,it could be used for analysis of the safety of novel cosmetic products.The present invention is also ideal for a long term monitoring of theeffects of compounds (up to 96 h), which therefore allows analysis ofthe longer-term toxicity than bacterial biosensors. The presentinvention is also suitable for use on different substrates such as solidand liquid supports.

Identification of Different Fungal Strains

It has been found that each particular compound produces a differentfingerprint when added to different fungal species. This can be used todiagnose the unknown fungus.

Procedure:

-   -   The fungus can be either transformed with the recombinant        aequorin gene or can be injected with the active aequorin.    -   Then this fungus can be subjected to a range of the antifungal        drugs, profiles of which have already been created.    -   Obtained profiles can be compared with the library of the        fingerprints and this way the fungal species can be identified.

FIGS. 22 a, b and c show that 5 known antifungal drugs (ketoconazole,clotrimazole, amphotericin B, nystatin and filipin) caused a different[Ca²⁺]_(c) response in 3 different species of Aspergillus (A. nidulans,A. niger, A. awamori).

Optimisation of the Current Antifungal Treatments

In order to administer drugs in the best possible way it is important todetermine no effect concentration and dose response curves, andfrequency for administration of drugs. Also, in view of the developingresistance of fungus and other eukaryotes to currently available drugs,clinicians are looking into using combination of drugs. The presentinvention is ideally suitable for such studies.

Identification of Compounds Which Would Prevent Fungal Growth onPlastics, Metals and Other Materials

Since the present invention is suitable for long term measurements it ispossible to monitor the development and growth of fungi on differentmaterials and plastics treated with different agents. It is possible tomonitor the state of fungal physiology by subjecting the organism todifferent physico chemical treatments and analysis of the profilesobtained.

Although the invention has been particularly shown and described withreference to particular examples, it will be understood by those skilledin the art that various changes in the form and details may be madetherein without departing from the scope of the present invention.

1. A method of determining the presence of a toxicant in a test sample, comprising the steps of; exposing a eukaryote that has been transformed with a light emitting Ca²⁺ regulated photoprotein gene to a test sample measuring the light produced by the transformed cell/organism determining whether the amount of light is above or below a defined threshold at the time of exposure.
 2. A method as in claim 1 wherein the eukaryote is a fungi.
 3. A method as in claim 2 wherein the fungi is a filamentous fungi.
 4. A method as in claim 2 wherein the fungi is of the Aspergillus species.
 5. A method as in claim 1 wherein the eukaryote is a mammalian cell.
 6. A method as in claim 1 wherein the eukaryote is a plant cell.
 7. A method as in claim 1 wherein the test sample comprises a toxicant.
 8. A method as in claim 1 wherein the light emitting Ca²⁺ regulated photoprotein gene is a recombinant gene.
 9. A method as in claim 1 wherein the light emitting Ca²⁺ regulated photoprotein gene is selected from the group comprising; aequorin gene halistaurin (mitrocomin) gene phialidin (clytin) gene obelin gene mnemiopsin gene berovin gene
 10. A method as in claim 1 wherein the light emitting Ca²⁺ regulated photoprotein gene may be a functional homologue of a gene selected from the group comprising; aequorin gene halistaurin (mitrocomin) gene phialidin (clytin) gene obelin gene mnemiopsin gene berovin gene
 11. A method as in claim 1 wherein the light emitting Ca²⁺ regulated photoprotein gene is an aequorin gene.
 12. A method as in claim 1 wherein the light emitting Ca²⁺ regulated photoprotein gene is a recombinant aequorin gene.
 13. A method as in claim 1 wherein the light that is measured is in the form of luminescence.
 14. A method as in claim 1 wherein the test sample is added in advance of the application of a stimulus to the test sample.
 15. A method as in claim 14 wherein the stimulus is at least one or more from the group comprising; mechanical perturbation, hypo-osmotic shock, change in external calcium chloride concentration, temperature shock and pH shock.
 16. A method as in claim 14 wherein the test sample is added 1 minute to 1 prior to the application of the stimulus.
 17. A method as in claim 14 wherein the test sample is added 5 prior to the application of the stimulus.
 18. A method as in claim 14 wherein the test sample is added 30 minutes prior to the application of the stimulus.
 19. A method of determining the presence of a toxicant in a test sample, comprising the steps of; exposing a eukaryote that has been transformed with a light emitting Ca²⁺ regulated photoprotein gene to a test sample measuring the light produced by the transformed cell/organism determining whether the amount of light is above a defined threshold at a specified time after the time of exposure.
 20. A method as in claim 19 which comprises the step of determining whether the amount of light is below a defined threshold.
 21. A method as in claim 19 wherein the specified time after the time of exposure is 11 minutes.
 22. A method as in claim 19 wherein the eukaryote is a fungi.
 23. A method as in claim 22 wherein the fungi is a filamentous fungi.
 24. A method as in claim 22 wherein the fungi is of the Aspergillus species.
 25. A method as in claim 19 wherein the eukaryote is a mammalian cell.
 26. A method as in claim 19 wherein the eukaryote is a plant cell.
 27. A method as in claim 19 wherein the test sample comprises a toxicant.
 28. A method as in claim 19 wherein the light emitting Ca²⁺ regulated photoprotein gene is a recombinant gene.
 29. A method as in claim 19 wherein the light emitting Ca²⁺ regulated photoprotein gene is selected from the group comprising; aequorin gene halistaurin (mitrocomin) gene phialidin (clytin) gene obelin gene mnemiopsin gene berovin gene
 30. A method as in claim 19 wherein the light emitting Ca²⁺ regulated photoprotein gene may be a functional homologue of a gene selected from the group comprising; aequorin gene halistaurin (mitrocomin) gene phialidin (clytin) gene obelin gene mnemiopsin gene berovin gene
 31. A method as in claim 19 wherein the light emitting Ca²⁺ regulated photoprotein gene is an aequorin gene.
 32. A method as in claim 31 wherein the light emitting Ca²⁺ regulated photoprotein gene is a recombinant aequorin gene.
 33. A method as in claim 19 wherein the light that is measured is in the form of luminescence.
 34. A method as in claim 19 wherein the test sample is added in advance of the application of a stimulus to the test sample.
 35. A method as in claim 34 wherein the stimulus is at least one or more from the group comprising; mechanical perturbation, hypo-osmotic shock, change in external calcium chloride concentration, temperature shock and pH shock.
 36. A method as in claim 34 wherein the test sample is added 1 minute to 1 prior to the application of the stimulus.
 37. A method as in claim 34 wherein the test sample is added 5 minutes prior to the application of the stimulus.
 38. A method as in claim 34 wherein the test sample is added 30 minutes prior to the application of the stimulus.
 39. A method of determining the presence of a toxicant in a test sample, comprising the steps of; exposing a eukaryote that has been transformed with a light emitting Ca²⁺ regulated photoprotein gene to a test sample measuring the light produced by the transformed cell/organism and comparing the light measurement data with a bank of known toxicity reference data.
 40. A method as in claim 39 wherein the method comprises the step of determining whether the amount of light is below a defined threshold.
 41. A method as in claim 39 wherein the specified time after the time of exposure is 11 minutes.
 42. A method as in claim 39 wherein the eukaryote is a fungi.
 43. A method as in claim 42 wherein the fungi is a filamentous fungi.
 44. A method as in claim 42 wherein the fungi is of the Aspergillus species.
 45. A method as in claim 39 wherein the eukaryote is a mammalian cell.
 46. A method as in claim 39 wherein the eukaryote is a plant cell.
 47. A method as in claim 39 wherein the test sample comprises a toxicant.
 48. A method as in claim 39 wherein the light emitting Ca²⁺ regulated photoprotein gene is a recombinant gene.
 49. A method as in claim 39 wherein the light emitting Ca²⁺ regulated photoprotein gene is selected from the group comprising; aequorin gene halistaurin (mitrocomin) gene phialidin (clytin) gene obelin gene mnemiopsin gene berovin gene
 50. A method as in claim 39 wherein, the light emitting Ca²⁺ regulated photoprotein gene may be a functional homologue of a gene selected from the group comprising; aequorin gene halistaurin (mitrocomin) gene phialidin (clytin) gene obelin gene mnemiopsin gene berovin gene
 51. A method as in claim 39 wherein the light emitting Ca²⁺ regulated photoprotein gene is an aequorin gene.
 52. A method as in claim 39 wherein the light emitting Ca²⁺ regulated photoprotein gene is a recombinant aequorin gene.
 53. A method as in claim 39 wherein the light that is measured is in the form of luminescence.
 54. A method as in claim 39 wherein the test sample is added in advance of the application of a stimulus to the test sample.
 55. A method as in claim 54 wherein the stimulus is at least one or more from the group comprising; mechanical perturbation, hypo-osmotic shock, change in external calcium chloride concentration, temperature shock and pH shock.
 56. A method as in claim 54 wherein the test sample is added 1 minute to 1 prior to the application of the stimulus.
 57. A method as in claim 54 wherein the test sample is added 5 minutes prior to the application of the stimulus.
 58. A method as in claim 54 wherein the test sample is added 30 minutes prior to the application of the stimulus.
 59. A method as in claim 39 wherein the method is used to determine the amount of toxicant in the sample.
 60. A method as in claim 39 wherein the method is used to identify the toxicant in the sample.
 61. A method of determining the presence of a toxicant in a test sample, comprising the steps of; exposing a eukaryote that has been transformed with a light emitting Ca²⁺ regulated photoprotein gene to a test sample measuring the light produced by the transformed cell/organism converting the light data into a cytosolic free calcium ion concentration trace, and comparing at least one parameter of the cytosolic free calcium ion concentration trace with a bank of known toxicity reference data.
 62. A method as in claim 61 wherein the method comprises the step of determining whether the amount of light is below a defined threshold.
 63. A method as in claim 61 wherein the eukaryote is a fungi.
 64. A method as in claim 63 wherein the fungi is a filamentous fungi.
 65. A method as in claim 63 wherein the fungi is of the Aspergillus species.
 66. A method as in claim 61 wherein the eukaryote is a mammalian cell.
 67. A method as in claim 61 wherein the eukaryote is a plant cell.
 68. A method as in claim 61 wherein the test sample comprises a toxicant.
 69. A method as in claim 61 wherein the light emitting Ca²⁺ regulated photoprotein gene is a recombinant gene.
 70. A method as in claim 61 wherein the light emitting Ca²⁺ regulated photoprotein gene is selected from the group comprising; aequorin gene halistaurin (mitrocomin) gene phialidin (clytin) gene obelin gene mnemiopsin gene berovin gene
 71. A method as in claim 61 wherein the light emitting Ca²⁺ regulated photoprotein gene may be a functional homologue of a gene selected from the group comprising; aequorin gene halistaurin (mitrocomin) gene phialidin (clytin) gene obelin gene mnemiopsin gene berovin gene
 72. A method as in claim 61 wherein the light emitting Ca²⁺ regulated photoprotein gene is an aequorin gene.
 73. A method as in claim 61 wherein the light emitting Ca²⁺ regulated photoprotein gene is a recombinant aequorin gene.
 74. A method as in claim 61 wherein the light that is measured is in the form of luminescence.
 75. A method as in claim 61 wherein the test sample is added in advance of the application of a stimulus to the test sample.
 76. A method as in claim 75 wherein the stimulus is at least one or more from the group comprising; mechanical perturbation, hypo-osmotic shock, change in external calcium chloride concentration, temperature shock and pH shock.
 77. A method as in claim 75 wherein the test sample is added 1 minute to 1 prior to the application of the stimulus.
 78. A method as in claim 75 wherein the test sample is added 5 minutes prior to the application of the stimulus.
 79. A method as in claim 75 wherein the test sample is added 30 minutes prior to the application of the stimulus.
 80. A method as in claim 61 wherein light is measured for between 1 minute and 5 hours following the application of the stimulus.
 81. A method as in claim 61 wherein light is measured for between 1 minute and 96 hours following the application of the stimulus.
 82. A method as in claim 61 wherein light is measured for 5 minutes following the application of the stimulus.
 83. A method as in claim 61 wherein the cytosolic free calcium ion trace is a plot of the cytosolic free calcium ion concentration against time.
 84. A method as in claim 61 wherein the parameter is at least one or more selected from the group comprising; lag time rise time absolute amplitude relative amplitude Length of transient at 20%, 50% and 80% of maximum amplitude number of cytosolic free calcium ion concentration increases percentage increase in final cytosolic free calcium ion concentration resting level percentage increase in recovery time percentage increase in pre-stimulating cytosolic free calcium ion concentration resting level total concentration of calcium
 85. A method as in claim 61 wherein the method is used to determine the amount of toxicant in the sample.
 86. A method as in claim 61 wherein the method is used to identify the toxicant in the sample. 